The principal objective of the work outlined in this proposal is to determine the mechanism of ATP hydrolysis by (Mg plus Ca)-dependent ATPase in red cell membranes. This enzyme functions as a Ca pump, using energy derived from the splitting of ATP to extrude Ca from the cell against a steep electrochemical gradient. We propose to investigate the mechanism of ATP hydrolysis by this enzyme in studies using terminally labeled 32P-ATP to label a phosphoenzyme intermediate in the reaction sequence of the enzyme. We will then determine whether activators of the enzyme (Mg, Ca, monovalent cations and a soluble protein or proteins in red cell hemolysates) act to stimulate formation or hydrolysis of this phosphoenzyme intermediate. We will also study the effect of N-ethylmaleimide, a potent inhibitor of the enzyme, on formation and hydrolysis of this phosphoenzyme. In addition we will attempt to identify and characterize a soluble protein activator of the enzyme which we have recently found to be present in membrane-free red cell hemolysates. BIBLIOGRAPHIC REFERENCES: Bond, G. H. and Hudgins, P. M.: Inhibition of ATPase activity in human red cell membranes by tetracaine. Biochem. Pharmacol. 25:267-270, 1976. Bond, G. H. and Hudgins, P. M.: Endogenous modifiers of human red cell membrane Ca-ATPase. Fed. Proc. 35:607, 1976.